The purpose of this study was to test if atrial natriuretic peptide (ANP) can prevent the mitochondrial permeability transition pore (mPTP) opening by inactivating glycogen synthase kinase 3β (GSK-3β). ANP prevented loss of mitochondrial membrane potential (ΔΨ m ) caused by H 2 O 2 in a dose-dependent manner. Similarly, cyclosporin A, an inhibitor of the mPTP opening, could also preserve ΔΨ m . ANP increased GSK-3β phosphorylation at Ser 9 , pointing to that ANP inactivates GSK-3β. ANP could not prevent the loss of ΔΨ m in cells transfected with the constitutively active GSK-3β (GSK-3β-S9A) mutant. The effects of ANP on GSK-3β phosphorylation and ΔΨ m were reversed by the selective PKG inhibitor KT5823 [2,3,9,10,11,12-hexahydro-10R-methoxy-2,9-dimethyl-1-oxo-9S,12R-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid, methyl ester]. In support, PKG markedly enhanced GSK-3β phosphorylation. ANP-induced GSK-3β phosphorylation was also abolished by the PI3K inhibitor LY294002 [2-(4-morpholinyl-4H-1-benzopyran-4-one hydrochloride)] and ANP could not prevent H 2 O 2 -induced loss of ΔΨ m in the presence of LY294002. These data suggest that ANP modulates the mPTP opening by inactivating GSK-3β through PKG and PI3K. GSK-3β is a common downstream target of PKG and PI3K. Prevention of the mPTP opening may underlie the mechanism for ANP’s protection against reperfusion injury.